This invention relates to a device for immersing a specimen in a cryogenic-fixation liquid for subsequent examination by means of a microscope and, in particular, by means of an electron microscope.
Cooling liquids at temperatures between -100.degree. C. and -190.degree. C. are used for numerous specimen-preparation operations. Such liquids are particularly used for the shock-freezing or cryogenic-fixation of biological or medical specimens for subsequent examination by means of a microscope, and, in particular, for examination by means of an electron microscope. The object of a cooling operation of this nature is to abstract as much heat as possible from the specimen within the shortest possible time. This object is particularly important in the case of biological or medical specimens which have not been pretreated (that is to say, which have not been subjected to a preliminary fixation and/or freezing-protection treatment), since, for these specimens, the cooling rate alone determines whether artificial separation of the water-rich plasmatic phases takes place which would render meaningful microscopic or histochemical examination impossible. The cooling rate determines whether the specimen freezes to a true-to-life, glassy form ("vitrification" takes place at cooling rates greater than 10,000.degree. C./sec.).
The high cooling rates of specimens which are required are obtained as a result of known injection procedures only in an extremely thin edge zone of the specimen, where the structure is, initially, well preserved and is cooled to below -80.degree. C. In contract, as a result of the poor heat-conduction capability of ice, zones which are situated deeper in the interior of the specimen, in particular inside comparatively large specimens with diameters in excess of 3 mm, generally remain, at temperatures which lie considerably above the critical limiting value of -80.degree. C. Complete stabilisation of the fine structures and low-molecular constituents of these specimens is, therefore, not effected. Portions of the injection apparatus, which is generally made of metal, likewise remain at a comparatively high temperature.
Inadequate cooling of a specimen could be countered by using cooling baths containing columns of liquid of sufficient height to permit the specimens to be moved, generally vertically, over distances of between 50 and 100 cm. By this means, an injection of a specimen at a velocity of 5 to 15 m/sec would be prolonged by a period of time ranging from 300 m/sec to 2 sec, which suffices, as a rule, for the complete cooling and stabilisation of specimens. Practical considerations such as the ease of handling and the safe operation of injection apparatus, have lead in practice to the depth of cooling baths being limited to approximately 10 cm. For an average injection velocity, the injection into a 10 cm. cooling bath is completed after only 100 m/sec. This period of time is sufficient for the vitrification of a thin edge zone, but is insufficient for the complete freezing of comparatively large specimens.
The specimen is necessarily brought to rest following the completion of the known injection operations, which leads very quickly, to heating of the edge zone of the specimen, after the edge zone has been cooled to temperatures near the original temperature of the cooling bath. The heating of the specimen is caused by the establishment of a temperature gradient in the cooling liquid. During this heating, a secondary change occurs in the frozen specimen as the limiting value of -80.degree. C. is exceeded, this change altering the previously stable condition.
If it is desired to cut specimens, in the amorphous/vitrified condition, on a cryogenic-microtome, and in particular on a cryogenic-ultramicrotome, a temperature of -140.degree. C. must not be exceeded in the edge zones, which cannot be guaranteed if the specimen suddenly comes to rest 100 msec after the beginning of the injection operation. In order to preserve the true-to-life vitrification initially obtained in the course of the rapid injection movement, it is necessary to use appropriate measures to restrict, to the greatest possible extent, the establishment of a temperature gradient which is formed as a secondary effect immediately after the specimen has come to rest, in the cooling liquid which is directly adjacent to the exposed surface of the specimen.